Project Summary: Mammalian skin and its appendages function as the outermost barrier of the body to protect inner organs from environmental hazards and keep essential fluids within the body. Homeostasis and integrity of mammalian skin are maintained by multiple progenitor and stem cell populations residing in distinct skin compartments such as basal cells in the interfollicular epidermis and bulge stem cells in hair follicles. In epithelial cells, cell adhesion, migration and proliferation are fundamental properties that are controlled by many mechanisms. Among key regulators, microRNAs (miRNAs) are a class of small, noncoding RNAs that take essential roles in mammalian gene regulation in diverse cell types and tissues. Despite modest regulation of individual targets, miRNAs broadly modulate a large number (60%) of genes and play important roles in a wide range of biological processes. In mammalian skin, the critical functions of the entire miRNA pathway in both embryonic skin development and maintenance of adult HF lineages have been well appreciated. In contrast, the knowledge of individual miRNAs for their targets and function remains scarce. Importantly, similar to other regulators, how miRNAs regulate cell migration and proliferation has not been examined in the context of intact skin in live animals. To address these important issues, we have developed techniques to directly capture miRNA and their targeted mRNA fragments and to image cell migration and proliferation as well as cytoskeleton dynamics in intact skin of live animals. Using these state-of-art tools together with our mouse models, we find that miR-205, the most highly expressed miRNA in epithelial stem cells, promotes cell migration by targeting components of adherens junctions, actin cytoskeleton and mechanosensing genes in both epidermis and hair follicles. In this project, we will further examine how miR-205-regulated cell migration alters the balance between epidermal proliferation and differentiation in the epidermis (Aim 1); how miR-205- induced cell migration triggers hair follicle growth in young and aged mice and how enhanced hair follicle growth affects hair follicle stem cells (Aim 2); and probe how the loss of Piezo1, a mechanically activated ion channel and a new miR-205 target, governs the quiescence of hair follicle stem/progenitor cells (Aim 3). Taken together, studies proposed here, if successful, will significantly enhance our knowledge about mechanisms mediated by individual miRNAs that govern cell migration and proliferation in live animals. The knowledge gained from these studies under normal and stressed conditions will pave the way to manipulate miRNAs and utilize epithelial stem cells for regenerative medicine.